PROJECT SUMMARY Mycoplasma genitalium (MG) is an emerging reproductive tract pathogen of significant public health concern. This sexually transmitted bacterium elicits a disease spectrum similar to Neisseria gonorrhoeae and Chlamydia trachomatis, yet may be more prevalent in certain clinical settings. Alarmingly, MG is becoming increasingly resistant to antibiotics with some infections totally untreatable with recommended regimens in the US. The recent FDA approval of an MG diagnostic test will certainly increase public awareness of MG, and demands for improved treatments. A hallmark of MG infection is long-term persistence despite the presence of specific antibodies in the genital tract. Over the past decade our laboratory has focused on the variability of the immunodominant MgpB and MgpC adherence proteins, and the local and systemic antibody response to these proteins during infection. We demonstrated that MgpB and MgpC undergo phase and antigenic variation via a unique system of homologous recombination between the mgpBC expression site and partial copies archived in the chromosome. We propose to extend these studies empowered by three recent advances: (1) the determination of the MgpC protein structure, which includes a sialic acid binding pocket, (2) our detailed analysis of antibody reactivity and antigenic variation in a collection of longitudinal specimens from MG-infected men, including evidence that the conserved C-terminal region of MgpC contains the dominant antigen targeted by patient antibodies, and (3) the discovery of the MG281 immunoglobulin binding protein whose role in pathogenesis is unexplored. MgpC C-terminal epitopes will be defined using engineered deletions and alanine substitutions analyzed by ELISA and surface plasmon resonance (SPR) to explore thermodynamics and kinetics of antibody binding. Complement killing and opsonophagocytosis assays will locate targets of bactericidal antibodies. Defining MgpC epitopes recognized by patient sera will inform improved serologic tests, essential to explore association of MG with serious upper reproductive tract sequelae in women. As the sialic acid binding pocket is embedded in the variable region of MgpC, we will measure changes in sialic acid binding affinity in different variants, and the effect of variant-specific antibodies on sialic acid binding and adherence. Understanding the role of MG281 in avoiding the biologic activity of anti-MG antibodies is a top priority in MG research especially as interactions of MG with innate immune effectors has not been defined. Therefore, our third aim will link the findings of Aims 1 and 2 to determine whether MG281 prevents killing by specific antibodies in sera of MG(+) men and in cervicovaginal exudates of MG(+) women. These experiments, along with our novel approaches pioneering experimental methods for the difficult field of mycoplasma research, will inspire improved prevention strategies to combat this increasingly antibiotic-resistant and important pathogen.